Docking Studies of Dipeptides to Metabotropic Glutamate Receptors

1. Kalyan C. Tirupula, Judith Klein-Seetharaman University of Pittsburgh School of Medicine Pittsburgh, PA SunandaSukumar, Benjamin Woo Albany College of Pharmacy Albany, NY 12208 N. Sukumar, Arshad S. Kokardekar Rensselaer Exploratory Center for Cheminformatics Research Department of Chemistry and Center for Biotechnnology and Interdisciplinary Studies Rensselaer Polytechnic Institute, Troy, NY 12180 Size Hyd Side 2E4U. Site 2: 340 78 189 CHAIN 1: PRO46, GLU49, LYS50, GLY51, THR52, GLY53, GLY58, ARG59, ILE60, ASN61, GLU62, ASP63, ARG64, GLN67, ARG68, SER100, ARG101, ASP102, SER149, TYR150, SER151, SER152, ALA172, SER173, THR174, SER218, GLU219, GLY220, ASP221, TYR222, GLY223, GLY248, ARG249, SER250, ASN251, ILE252, ARG253, TYR256, ARG277, SER278, ASP279, ASP280, ARG282, GLU283, ALA286, ARG290, ASP301, GLY302, GLN306, GLU307, SER308, ILE309, LYS311, PHE337, PHE354, GLN357, LYS358, GLN360, ILE379, SER381, ASN383, TYR384, GLU385, LYS389, LYS389, LYS477, TYR478, SER479. Total Number of Residues: 68 Total Number of Residues in site 4 of Human mGluR6: 44 Total Number of Common Residues: 35 mGluR I mGluR II mGluR III mGluR 3 mGluR 1 mGluR 5 mGluR 2 Couple to Gq proteins to activate phospholipase C Couple to Gi/o proteins to negatively regulate adenylyl cyclase activity mGluR 4 mGluR 6 mGluR 7 mGluR 8 Autoreceptors coupled to Gi/o proteins to decrease adenylyl cyclase activity Docking Studies of Dipeptides to Metabotropic Glutamate Receptors • Introduction • Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system whose actions are regulated by ionotropic and metabotropic glutamate receptors (mGluR). • mGluRs belong to the G protein coupled receptor (GPCR) family and are important potential drug targets. • mGluRs have been implicated in different brain functions and dysfunctions including learning, memory, pain perception, neurodegeneration, schizophrenia and addiction. • Mutations in mGluR6 are associated with night blindness but the actual function of mGluR6 is yet unknown. mGluR6 mGluR6 expression and purification. Shown in the picture is a western blot verifying the expression of mGluR6 with C-terminal 1D4 tag. mGluR6 The top docked poses of Gly-Glu with the homology model of human mGluR6 from 2E4U in FlexX. Glutamate Receptors Cyclo Gly-Glu(eq) with protein Ionotropic Glutamate Receptors (iGluR) (Control Activation state of neurons) Cyclo Gly-Glu(ax) with protein Metabotropic Glutamate Receptors (mGluR) (Glutametergic neurotransmission modulator) Size Hyd Side 2E4U. Site 2: 340 78 189 CHAIN 1: PRO46, GLU49, LYS50, GLY51, THR52, GLY53, GLY58, ARG59, ILE60, ASN61, GLU62, ASP63, ARG64, GLN67, ARG68, SER100, ARG101, ASP102, SER149, TYR150, SER151, SER152, ALA172, SER173, THR174, SER218, GLU219, GLY220, ASP221, TYR222, GLY223, GLY248, ARG249, SER250, ASN251, ILE252, ARG253, TYR256, ARG277, SER278, ASP279, ASP280, ARG282, GLU283, ALA286, ARG290, ASP301, GLY302, GLN306, GLU307, SER308, ILE309, LYS311, PHE337, PHE354, GLN357, LYS358, GLN360, ILE379, SER381, ASN383, TYR384, GLU385, LYS389, LYS389, LYS477, TYR478, SER479. Total Number of Residues: 68 Total Number of Residues in site 4 of Human mGluR6: 44 Total Number of Common Residues: 35 Size Hyd Side 2E4U. Site 2: 340 78 189 CHAIN 1: PRO46, GLU49, LYS50, GLY51, THR52, GLY53, GLY58, ARG59, ILE60, ASN61, GLU62, ASP63, ARG64, GLN67, ARG68, SER100, ARG101, ASP102, SER149, TYR150, SER151, SER152, ALA172, SER173, THR174, SER218, GLU219, GLY220, ASP221, TYR222, GLY223, GLY248, ARG249, SER250, ASN251, ILE252, ARG253, TYR256, ARG277, SER278, ASP279, ASP280, ARG282, GLU283, ALA286, ARG290, ASP301, GLY302, GLN306, GLU307, SER308, ILE309, LYS311, PHE337, PHE354, GLN357, LYS358, GLN360, ILE379, SER381, ASN383, TYR384, GLU385, LYS389, LYS389, LYS477, TYR478, SER479. Total Number of Residues: 68 Total Number of Residues in site 4 of Human mGluR6: 44 Total Number of Common Residues: 35 No Ligand L-Glu L-Gly L-Gln Gly-Glu Conclusions • Addiction & Withdrawal • Focus of this work is the putative role of mGluRs in addiction. • mGluR6 & mGluR8  implicated in heroin addiction. • Group II mGluRs  nicotine addiction in animal models. • Specific role of mGluRs in addiction has not been firmly established, and a systematic analysis of the role of different subtypes has not been carried out. • Recently a genetic linkage study associated tolerance to heroin addiction with alleles in proximity to the mGluR6 gene1. • Several animal model studies of nicotine, alcohol and cocaine dependence implicated mGluRs in inhibiting dependence and alleviating withdrawal symptoms. • The dipeptide glycine-glutamine (Gly-Gln; derived from β-endorphin) is a known modulator of addiction to a variety of drugs. • Millington et al*. have shown that rats simultaneously administered nicotine and Gly-Gln were found to be less susceptible to nicotine addiction than rats administered nicotine alone. • Similar experiments have been carried out in rats with morphine and alcohol addiction. • The actual target(s) for Gly-Gln are not known. • Hypothesis  mGluR, bind and are activated by Gly-Gln. • This hypothesis would have important implications in the treatment of addiction, as mGluRs are well studied due to their involvement in a variety of neuronal disorders, and different negative and positive modulators are known. • These modulators bind in the transmembrane domain and their effects on receptor activity correlate well with binding to active and inactive models of the receptors. • The order of binding of ligands on 1EWT was cyclo Gly-L-Gln < Gly-D-Gln < Gly-L-Gln • The order of binding of ligands on the homology model of Human mGluR6 was Gly-s-Glu < Gly-r-Glu < Cyclo Gly-Glu(ax) < Cyclo Gly-Glu(eq) • Gly-Glu activates mGluR6  Result from Functional Assay mGluR1 The alpha sites for Gly-Gln in the structure of the extracellular domain of mGluR1 (1EWT) in MOE. Future Work • To predict the binding sites and specificity of Gly-Glu, Gly-Gln binding to different subtypes of mGluRs: The generally conserved extracellular domain in the mGluR family of GPCRs suggest that binding may also occur in other mGluRs. We will test this hypothes and investigate if there would be differences expected for different subtypes of mGluRs. • To determine the co-crystal structures of Gly-Glu, Gly-Gln and other dipeptides bound to various families of mGluRs: The resulting structures will be compared to docking results. • To experimentally validate the results from Aims 1 & 2 for mGluR1/6: We will also investigate the effects of allosteric modulators of mGluRs on Gly-Gln mediated activation. • Follow-up rat experiments on morphine and heroin addiction are planned. size hyd side 1EWK site 4: 120 27 62 2:(TYR74,TRP110,GLY163,SER164,SER165,SER166,SER186,ALA187,THR188,SER189,ASP208,GLU233,GLY234,ASN235,TYR236,GLU292,GLY293,MET294,ASP318,GLY319,TRP320,ALA321,ARG323,LYS341,LEU342,GLN343,SER344,SER408,LYS409,PHE412) *total 30 1EWT site 4: 178 43 99 2:(TYR74,ARG78,TRP110,GLY163,SER164,SER165,SER166,ILE169,SER186,ALA187,THR188,SER189,ASP191,LEU192,ASP208,ASN235,TYR236,SER263,GLU292,GLY293,MET294,ASP318,GLY319,ALA322,ARG323,GLU325,ILE340,LYS342,SER344,PRO345,GLU346,ASP351,TYR404,VAL405,GLN406,ASP407,SER408,LYS409,PHE412) *total 39 23 residues common 1EWK site 7: 108 25 66 1:(HIS55,GLN56,ARG71,TYR74,TARG78,TRP110,GLY163,SER164,SER165,SER186,ALA187,THR188,SER189,TYR236,SER263,ASN264,GLU292,GLY293,MET294,ASP318,GLY319,ARG323,LYS409) *total 23 1EWT site 3: 204 49 114 1:(GLN56,ARG71,GLN73,TYR74,ARG78,TRP110,HIS111,SER112,GLY163,SER164,SER165,SER166,VAL167,SER186,ALA187,THR188,ASP208,THR232,GLU233,GLY234,ASN235,TYR236,GLY237,TYR262,SER263,ASN264,GLU292,GLY293,MET294,ASP318,GLY319,ALA321,ASP322,ARG323,GLU325,ILE340,LYS341,LEU342,GLN343,SER344,PRO345,GLU346,VAL405,GLN406,ASP407,SER408,LYS409,PHE412,ASP480) *total 49 21 residues common We thank Professor Curtis Breneman & Professor William Millington for helpful discussions. Funding provided by NIH Molecular Libraries Roadmap Initiative Grant # 1P20HG003899-01. * Goktalay, G; Cavun, S; Levendusky, M. C; Hamilton, J. R; Millington, W. R. Glycyl-glutamine inhibits nicotine conditioned place preference and withdrawal, Eur. J. Pharmacol. 2006(530), 95-102. http://reccr.chem.rpi.edu/